PCL-coated hydroxyapatite scaffold derived from ...

Materials Science and Engineering C 42 (2014) 264–272

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PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: In vitro cell culture studies Dajana Milovac a,⁎, Tatiana C. Gamboa-Martínez b, Marica Ivankovic a, Gloria Gallego Ferrer b,c, Hrvoje Ivankovic a a b c

Faculty of Chemical Engineering and Technology, University of Zagreb, Croatia Centre for Biomaterials and Tissue Engineering, Universitat Politècnica de València, Spain Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine, Valencia, Spain

a r t i c l e

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Article history: Received 11 March 2014 Received in revised form 14 April 2014 Accepted 7 May 2014 Available online 22 May 2014 Keywords: Cuttlefish bone Poly(ε-caprolactone) Hydroxyapatite scaffold Tissue engineering Cell differentiation

a b s t r a c t In the present study, we examined the potential of using highly porous poly(ε-caprolactone) (PCL)-coated hydroxyapatite (HAp) scaffold derived from cuttlefish bone for bone tissue engineering applications. The cell culture studies were performed in vitro with preosteoblastic MC3T3-E1 cells in static culture conditions. Comparisons were made with uncoated HAp scaffold. The attachment and spreading of preosteoblasts on scaffolds were observed by Live/Dead staining Kit. The cells grown on the HAp/PCL composite scaffold exhibited greater spreading than cells grown on the HAp scaffold. DNA quantification and scanning electron microscopy (SEM) confirmed a good proliferation of cells on the scaffolds. DNA content on the HAp/PCL scaffold was significantly higher compared to porous HAp scaffolds. The amount of collagen synthesis was determined using a hydroxyproline assay. The osteoblastic differentiation of the cells was evaluated by determining alkaline phosphatase (ALP) activity and collagen type I secretion. Furthermore, cell spreading and cell proliferation within scaffolds were observed using a fluorescence microscope. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Composites of bioactive ceramics and biodegradable polymers have been proven to be attractive scaffold materials for use in bone tissue engineering [1–4]. An ideal scaffold should mimic both the structure and mechanical properties of the natural bone. It should provide a highly porous matrix with interconnected pores that enables the transport of nutrients, oxygen and metabolic waste products. Its surface properties must be suitable for cell adhesion, proliferation and differentiation. Also, the scaffold should be bioresorbable with a controllable degradation rate to match the replacement by new tissue. Additionally, the scaffold should possess sufficiently high mechanical properties such as stiffness, strength and toughness. Among calcium phosphate-based ceramics hydroxyapatite (HAp), Ca10(PO4)6(OH)2, has received particular attention in recent years due to its chemical similarity to the inorganic matrix of natural bone, excellent osteoconductivity and bioactivity [5, 6]. The major drawback of the HAp scaffolds is their poor mechanical

⁎ Corresponding author at: Dept. of Inorganic Chemical Technology and Non-Metals, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev trg 20/I, HR-10000 Zagreb, Croatia. Tel.: +385 1 4597 226; fax: +385 1 4597 260. E-mail address: [email protected] (D. Milovac).

http://dx.doi.org/10.1016/j.msec.2014.05.034 0928-4931/© 2014 Elsevier B.V. All rights reserved.

properties, especially the brittleness and low fracture toughness. Therefore, they cannot be used in load bearing applications. To overcome these disadvantages HAp has been combined with polymers that provide flexibility to the brittle system. Due to its biodegradability, biocompatibility, appropriate mechanical properties, and low emission of harmful byproducts, polycaprolactone (PCL) has been widely used to prepare HAp/PCL composite scaffolds. Currently, porous HAp scaffolds have been prepared by a number of manufacturing techniques including polymer foam replication, sol–gel and freeze casting, solid freeform fabrication etc. [7–13]. Natural resources basically comprised of calcium carbonate (corals, seashells, nacres, cuttlefish) are receiving growing interest because of their possible conversion to HAp via a hydrothermal reaction. HAp prepared from natural sources is nonstoichiometric, and has other ions incorporated, mainly CO23 −, and traces of Na+, Mg2+, Fe2+, F−, and Cl− [14]. Carbonated HAp is closer to the chemistry of natural human bone than stoichiometrically pure HAp [15] and has been shown experimentally to have enhanced biocompatibility [16,17]. In recent years, a number of studies on the conversion of natural aragonite (CaCO3) structures to HAp have been reported [18–28]. Of these, cuttlefish bone has been the most extensively studied [21–28]. Rocha and co-workers [21–24] were the first who performed the hydrothermal transformation of aragonitic cuttlefish bone to produce hydroxyapatite scaffolds retaining the cuttlebone

D. Milovac et al. / Materials Science and Engineering C 42 (2014) 264–272

architecture. The studies included tests for biocompatibility of the powdered scaffolds with osteoblasts and in vitro bioactivity of the scaffolds. Battistella et al. [27] performed biological characterization of hydroxyapatite scaffolds derived from cuttlefish bone using the preosteoblastic cell line MC3T3-E1. In our recent publication [26] we reported on preparation and physical–chemical characterization of the PCL-coated highly porous hydroxyapatite scaffold derived from cuttlefish bone. First, hydrothermal transformation (HT) of aragonitic cuttlefish bone into hydroxyapatite was performed at 200 °C retaining the cuttlebone architecture. Then, the scaffold was coated with the PCL using the vacuum impregnation technique. PCL coating on HAp was found to be very effective in increasing the mechanical properties of the scaffold. The compressive strength (0.88 MPa) and the elastic modulus (15.5 MPa) of the HAp/PCL composite scaffold were within the lower range of properties reported for human trabecular bones. The in vitro mineralization of calcium phosphate (CP) that produces the bone-like apatite was observed on both the pure HAp scaffold and the HAp/PCL composite scaffold. Similar PCL reinforced HAp scaffolds were recently reported by Kim et al. [28] in which human osteoblast-like MG63 cells adhered well, proliferated and showed higher expression of osteogenic markers (ALP, Runx2 and Col1α1) than pure HAp after 5 days of culture. This work aimed at investigating the in vitro cell viability of our HAp/ PCL scaffold and its potential as a substrate for bone regeneration, as a prerequisite to address its in vivo feasibility. MC3T3-E1 preosteoblast cells were cultured on the surface of the HAp/PCL scaffold. Its viability and proliferation were monitored by DNA quantification and Live/ Dead test and its differentiation potential by total collagen content, ALP activity and immunofluorescence of collagen type I. Our study addresses longer culture times than in the Kim [28] assay, 21 days, where cell differentiation is higher than at shorter times. Furthermore, we show that PCL coating does not obstruct pore connectivity and cells seeded on the surface of the scaffold are capable of invading the entire construct after 21 days in culture, which is very important for the future in vivo osseointegration of the construct. 2. Materials and methods 2.1. Hydrothermal synthesis of porous hydroxyapatite Cuttlefish bones (Sepia officinalis L.) from the Adriatic Sea were used as starting material for the hydrothermal synthesis of hydroxyapatite (HAp). The bones were carefully cut into small pieces (2 cm3) and treated with an aqueous solution of sodium hypochlorite (NaClO, 13% active chlorine, Gram-mol) for 12 h at room temperature to remove the organic component. The pieces of cuttlefish bone were then sealed with the required volume (respecting the molar ratio of Ca/P = 1.67) of a 0.6 M aqueous solution of ammonium dihydrogenphosphate (NH4H2PO4, 99%, Scharlau) in a TEFLON lined stainless steel pressure vessel at 200 °C for 72 h. The pressure inside the reactor was selfgenerated by water vapor and reached 18 bar. After hydrothermal treatment the resulting pieces of HAp scaffold were washed with boiling demineralized water and dried at 105 °C. 2.2. Preparation of the HAp/PCL composite scaffold HAp scaffolds were impregnated with a PCL solution in chloroform using the vacuum impregnation unit (CitoVac, Struers). The purpose of impregnation in a vacuum is to remove the air out of the pores, thus ensuring that the PCL solution can easily flow into the pores. A homogenous 20% (w/v) solution of poly(ε-caprolactone) (PCL, Mn = 45,000, Sigma-Aldrich) was prepared by intensive stirring of PCL pellets in chloroform (CHCl3, p.a., Kemika) and poured into a cup connected to the vacuum chamber through the tube. The specimens of porous HAp were put in a beaker and placed in the vacuum chamber. The pressure

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was set to 0.11 bar. After 10 min the valve was open to suck the PCL solution through the tube filling the beaker over the porous specimens. The specimens were soaked for 10 min. Then, the vacuum was stopped to allow the air pressure to force the PCL solution into the pores in the specimens. The beaker was removed from the vacuum chamber. The soaked scaffolds were put on a net and placed in the vacuum chamber again. The vacuum was restored in order to remove the excess PCL solution away from the scaffolds and to dry the specimens. 2.3. Scaffolds in vitro cell culture Each scaffold was cut into quadratic shape (7 mm × 7 mm × 1 mm) and sterilized by gamma radiation with a dose of 26 kGy. Specimens were repeatedly washed in Dulbecco's phosphate buffered saline, DPBS (Sigma-Aldrich) and preconditioned in Dulbecco's Modified Eagle's Medium, DMEM (Invitrogen) containing 1 g/l glucose supplemented with 10% fetal bovine serum, FBS (Invitrogen), and 1% penicillin/streptomycin, P/S (100 units/ml/100 mg/ml, Lonza) overnight. Mouse preosteoblastic cells, MC3T3-E1 (obtained from RIKEN CELL BANK, Japan) were expanded into 75 ml culture flasks and grown in DMEM supplemented with 10% FBS and 1% P/S at 37 °C and 5% CO2. After reaching confluence, MC3T3-E1 cells were trypsinized (0.25% trypsin/EDTA solution, Sigma Chemical, USA) from the culture flask and counted with a hemocytometer. Each scaffold was transferred into the respective well of a 24 well plate and seeded onto their surface in a drop wise manner at a cell density of 2 × 104 cells/scaffold. To promote cellular adhesion samples were incubated for 1 h and afterwards DMEM supplemented with FBS. After three days of culture 1% ascorbic acid (Sigma-Aldrich) and 1% β-glycerophosphate (Sigma-Aldrich) were added to the medium to promote osteogenic differentiation for the total 21 days of culture. The culture media were renewed every three days and triplicates of each type of scaffold were used. A glass cover was used as control for each time of culture. 2.4. Scanning electron microscope observation The morphology of HAp and HAp/PCL composite scaffolds before the culturing and after 21 days of culture was examined. Previously the samples seeded with cells were carefully washed with DPBS and fixed in 2.5% glutaraldehyde solution at 4 °C for 1 h. The samples were dehydrated in ethanol-graded solutions (30%, 40%, 60%, 70%, 80%, 96% and 100%) for 5 min each and dried at room temperature overnight. All dried samples were sputter coated with gold and examined by SEM (JEOL JSM 5410) at a voltage of 15 kV. 2.5. Viability assay Cell viability was determined by staining cells using a Live/Dead® viability/cytotoxicity Kit (Invitrogen). After 4 and 10 days of culture the samples were washed in DPBS and then incubated with 2 μM of calcein and 4 μM of ethidium homodimer-1 (EthD-1) for 15 min at 37 °C in a humidified atmosphere under 5% CO2. Live cells were stained in green due to the enzymatic conversion of the nonfluorescent, cellpermeant dye calcein AM to the intensely fluorescent calcein. Dead cells are identified by staining with EthD-1, which enters cells with damaged membranes, and upon binding to nucleic acids produces a bright red fluorescence. The samples were observed by a fluorescence microscope Nikon Eclipse 80i. 2.6. Cellular proliferation and DNA quantification MC3T3-E1 proliferation on the scaffolds was determined using a fluorimetric PicoGreen dsDNA quantification Kit (Invitrogen) following the manufacturer's protocol. After removing the medium from the culture wells at days 0, 3, 7, 14 and 21 the samples were kept at −80 °C for further analysis. Prior to DNA quantification, samples were thawed,

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washed with 1 ml of DPBS and then digested with 1 ml of 50 mg/ml proteinase K (Roche) in 100 mM K2HPO4 (pH 8.0) at 56 °C in a water bath overnight. The samples were sonicated for 30 min, vortexed and centrifuged at 10,000 rpm for 5 min at 4 °C. Enzymatic reaction was inactivated at 96 °C for 10 min. The concentration of DNA in the samples was determined from a calibration curve of lambda DNA standards. The sample fluorescence was measured using a microplate reader VICTOR 3 (Perkin-Elmer) at 480/525 nm.

2.7. Observation of cell distribution into the scaffold by fluorescence microscopy In order to examine additionally the cell proliferation on the scaffolds and cell distribution inside the scaffold the samples obtained after 14 and 21 days of culture were processed for fluorescence microscopy observation. The medium from the 24-well plate was removed and the samples were washed with DPBS and fixed in formalin for 1 h at 4 °C. Afterwards, the samples were pre-incubated with permeabilization buffer (10.3 g sucrose, 0.292 g sodium chloride, 0.06 g magnesium chloride, 0.476 g HEPES buffer, and 0.5 ml Triton X, in 100 ml water, pH 7.2) for 5 min at room temperature followed by DPBS washing. Incubation with blocking buffer for 30 min at room temperature and counterstaining with ethidium homodimer for 30 min were performed. The samples were then washed in DPBS and gently cut with a scalpel. Images from stained constructs were obtained using a fluorescence microscope Nikon Eclipse 80i.

2.10. Immunofluorescence assay for collagen type I To study the presence of the extracellular matrix (ECM) on the scaffolds, immunofluorescence staining of collagen type I was performed at 3 and 14 days. Prior to immunostaining, the cultured samples were fixed with formalin at 10% for 1 h and 4 °C and then washed 3 times in DPBS. Thick sections of 20 μm were used to analyze the inner structure. The immunostaining was carried out following standard protocols. Samples were rinsed with DPBS and the permeabilization of the cell membrane was performed with buffer (10.3 g sucrose, 0.292 g sodium chloride, 0.06 g magnesium chloride, 0.476 g HEPES buffer, and 0.5 ml Triton X, in 100 ml water, pH 7.2) for 5 min at room temperature. The sample slices were incubated with a rabbit antimouse collagen I (1:50, ABCAM) in a 1% solution of bovine serum albumin (BSA) in DPBS at room temperature for 1 h. Followed by incubation for 1 h with the secondary antibody cy3 conjugated goat antirabbit (1:200, Jackson ImmunoResearch) in a 1% solution of BSA in DPBS at room temperature. Finally, the nuclei were stained with 0.2 μg/ml of 4′,6-diamidino-2phenylindole (DAPI). Immunofluorescent analyses were performed in triplicate with a fluorescence microscope Nikon Eclipse 80i. 2.11. Statistical analysis In this study all the experimental groups were carried out in triplicate and the results are presented as average ± standard deviation and were analyzed using a one factor ANOVA statistical study. Differences were considered significant if the p value was less than 0.05. 3. Results

2.8. ALP activity 3.1. Scanning electron microscopy The activity of alkaline phosphatase (ALP) was evaluated by measuring the conversion of p-nitrophenylphosphate (pNPP) to p-nitrophenol as the result of the activity of a cellular extract [29]. After culturing for 0, 3, 7, 14 and 21 days, the samples were repeatedly washed with DPBS and then fragmented and dispersed in 200 μl of lysis buffer (0.2% Triton X-100, 10 mM Tris–HCl pH 7.2) on ice for 10 min, and further sonicated for 2 min. Afterwards the samples were centrifuged for 7 min at 14,000 rpm at 4 °C. 100 μl of each cell lysate solution was added to 100 μl of pNPP substrate and incubated in the dark at 37 °C for 2 h. After the incubation, the reaction was quenched by adding 50 μl of 0.1 M NaOH to each well. The solution was transferred to a cuvette and absorbance at 405 nm was measured using the Perkin-Elmer VICTOR 3 microplate reader. The amount of ALP was then calculated against a standard curve of pNPP.

Fig. 1(a–d) shows SEM images of the macroporous HAp and HAp/ PCL scaffolds, respectively, before the cell culturing. As observed, the interconnected structure of the cuttlefish bone is maintained after the hydrothermal conversion into HAp (Fig. 1(b)) as well as after polymer impregnation (Fig. 1(d)). In Fig. 2, SEM micrographs of the surface of the HAp and the HAp/PCL scaffolds seeded with MC3T3-E1 cells after 21 days of culturing are given. As seen from the low magnification images of Fig. 2(a) and (c) the surface of both scaffolds was almost entirely covered by the cells and the extracellular matrix (ECM) they secreted. Most of the pores at the surface of the HAp/PCL scaffold are covered by a layer of cells and matrix. It is difficult to extract conclusions about the morphology of the cells. 3.2. Cell viability

2.9. Total collagen content determination Hydroxyproline content was measured using an adapted protocol of Kafienah and Sims [30]. In brief, proteinase K digested samples (digestion described above) were hydrolyzed in concentrated HCl (38%) at 110 °C for 18 h. After cooling at room temperature and centrifugation the samples were dried in a heating block at 50 °C. When evaporation was complete the hydrolysate was dissolved in 200 μl of ultra-pure H2O and 60 μl of this solution was placed into the wells of a 96 well plate (in triplicate). To each well, 20 μl of freshly prepared assay buffer and 40 μl of chloramine-T reagent were added. Following a 20 min incubation at room temperature, 80 μl of 4-(dimethylamino) benzaldehyde solution (6 ml isopropanol (Scharlab), 3 ml 70% perchloric acid (Sigma), and 4.5 g 4-(dimethylamino) benzaldehyde (Sigma)) were carefully added. The microplates were incubated for 20 min at 50 °C in an oven and subsequently cooled down. Absorbance of each sample was measured at 570 nm with the Perkin-Elmer VICTOR 3 microplate reader and the hydroxyproline content of the samples was calculated using a standard curve.

The Live/Dead assay, a two-part dye staining live cells green and dead cells red, has been used to study cell viability on both the HAp and HAp/PCL scaffolds for 4 and 10 days. From the fluorescence microscopy images given in Fig. 3 it can be observed that MC3T3-E1 cells proliferated and remained viable after 10 days on both substrates. At 10 days, the cell density on both scaffolds was much higher than that at 4 days. Dead cells were present in low numbers as detected by the low bright red fluorescence. The cells grown on the HAp/PCL composite scaffold exhibited greater spreading than cells grown on the HAp scaffold. 3.3. Cell proliferation by DNA quantification Cell proliferation studies were performed at different time points over a period of 21 days. As seen from Fig. 4, the DNA content increased on HAp scaffold at each time point until day 14 and on the HAp/PCL scaffold until day 21. Following day 14, DNA content remained statistically unchanged on the HAp scaffold resulting in typical sigmoidal curve.


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Fig. 1. SEM images of the macroporous HAp (a), the cross-section of HAp scaffold showing channels formed by convoluted pillars (b), HAp/PCL scaffold (c) showing maintained pore interconnectivity after PCL impregnation (d).

Fig. 2. SEM micrographs of the surface of the HAp (a,b) and the HAp/PCL scaffolds (c,d) seeded with MC3T3-E1 cells after 21 days of culturing.

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Fig. 3. Fluorescence microscopy images of stained MC3T3-E1 cells after culturing for 4 and 10 days on the HAp and HAp/PCL scaffolds. Calcein AM stained live cells green and ethidium homodimer-1 stained the nuclei of dead cells red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.4. Cell distribution into the scaffold by fluorescence microscopy MC3T3-E1 cell distribution on the HAp and the HAp/PCL scaffolds was analyzed after 14 and 21 days of culturing by a fluorescence microscope. Also, the cell colonization inside the HAp/PCL composite scaffold was examined after 21 days of culturing. As seen from Fig. 5 after 14 days of culture the cells proliferated on the surface and within the pores of the HAp/PCL scaffold which appeared to be more colonized by cells with partial or even complete closure of some pores, in comparison to the pores of the HAp scaffold.

It is very interesting to note that PCL coating in the walls of the HAp scaffold did not affect its pores connectivity. Consequently, fluorescence micrographs of the HAp/PCL scaffold inner regions (Fig. 6) revealed that the penetration of cells occurred through the entire depth of the scaffold over the experimental period of 21 days, despite the fact that seeding was done in one of the external surfaces. 3.5. Alkaline phosphatase activity The activity of intracellular alkaline phosphatase (ALP) of MC3T3-E1 cells cultured on HAp and HAp/PCL was monitored at 3, 7, 14, and 21 days as shown in Fig. 7, as an indication of osteoblastic differentiation. ALP activity of the cells grown on HAp and HAp/PCL scaffolds increased with the increase of culture time between days 3 and 14 reaching a maximum on day 14. An increase in bone specific ALP activity followed by a significant decrease at day 21 showed that the preosteoblasts seeded onto the scaffolds undergo a differentiation process into the direction of a mature phenotype. 3.6. Total collagen content

Fig. 4. The DNA content of MC3T3-E1 preosteoblasts cultured on HAp and HAp/PCL scaffolds after 0, 3, 7, 14 and 21 days in culture. The values are represented as the mean ± standard deviation of three replicates.

Total collagen content (Fig. 8) within the HAp and HAp/PCL was determined at different time points from the hydroxyproline amount after acid hydrolysis and reaction with chloramine-T and 4-(dimethylamino) benzaldehyde. Total collagen contents within the HAp and HAp/PCL steadily increased with time in culture, in a statistically significant manner indicating the formation of a new extracellular matrix (ECM) within the scaffolds. At day 3 no collagen was detected within the HAp/PCL scaffold. At day 7 total collagen content within the HAp scaffold was significantly higher compared with the HAp/PCL scaffold. At days 14 and 21 in


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Fig. 5. Fluorescence micrographs of MC3T3-E1 cells cultured on the HAp and HAp/PCL composite scaffolds after 14 and 21 days of culturing.

culture a significant higher level of collagen production was observed within the HAp/PCL scaffold compared with the HAp scaffold. 3.7. Immunofluorescence assay of collagen type I To confirm the osteoblastic differentiation on the HAp/PCL scaffold immunofluorescent staining of collagen type I was used. At day 3 (Fig. 9(a)) collagen type I was expressed at a relatively low level and fluorescence pictures show sparse dots around some cells. At day 21 (Fig. 9(b)) collagen type I produced strong fluorescence intensity, suggesting abundant secretion around all cells within the HAp/PCL scaffold.

In our previous studies [25,26] interconnected highly porous HAp and HAp/PCL scaffolds were prepared using very simple and inexpensive methods. First, hydrothermal transformation (HT) of aragonitic cuttlefish bone into hydroxyapatite was performed at 200 °C retaining the cuttlebone architecture. Previous reported results [26] have shown that the removal of the organic component from the cuttlefish bone and the HT transformation of aragonitic cuttlefish bone into HAp had a negative effect on the mechanical properties of the scaffold. The HAp scaffold was much more fragile with low and irregular resistance of its lamellae to applied loads as compared to the raw cuttlefish bone scaffold. It was hypothesized that the role of the organic component

4. Discussion Bone tissue engineering requires porous threedimensional (3D) scaffolds that can provide a framework for the cells to adhere, proliferate, and create an extracellular matrix.

Fig. 6. Fluorescence micrograph showing MC3T3-E1 invasion in the HAp/PCL composite scaffold after 21 days of culturing. The image presents the entire cross-section of the scaffold.

Fig. 7. ALP activity of MC3T3-E1 cells cultured on HAp and HAp/PCL at different time points. The values are represented as the mean ± standard deviation of three replicates.

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Fig. 8. Total collagen content determined in HAp and HAp/PCL composite scaffolds. The values are represented as the mean ± standard deviation of three replicates. The * indicates significant difference (p b 0.05) in the same sample at different times of culture and the ** denotes significant difference between the samples on the same culture day.

(β-chitin) in the raw cuttlefish bone scaffold was to redistribute compressive stress and dissipate energy during deformation along the organic layer, rather than through the inorganic crystals, inhibiting propagation of cracks. The similar role was expected from PCL coating on the HAp scaffold. The scaffold was coated with the PCL using vacuum impregnation technique. It was found [26] that PCL coating on HAp was very effective in increasing the mechanical properties of the scaffold which is one requirement for bone regeneration. The HAp/PCL composite scaffold displayed the highest compressive strength (0.88 MPa) and the elastic modulus (15.5 MPa) compared to the raw cuttlefish

Fig. 9. Immunofluorescent staining images of collagen type I (red) by MC3T3-E1 cells cultured at 3 (a) and 21 (b) days in the HAp/PCL scaffold. DAPI stained the cellular nuclei blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(0.46 MPa; 6.2 MPa) and the uncoated HAp scaffold (0.15 MPa; 0.7 MPa). The applied vacuum infiltration of PCL probably facilitates the filling of the crack-like defects inhibiting crack propagation on HAp scaffolds' surface. In addition, interconnectivity, as shown by the scaffolds examined in this work is especially important for the cells and tissue to penetrate the interior of the porous scaffolds and as the pathways for the transportation of biofluids, nutrients and metabolic wastes. Cell viability and proliferation on the scaffolds is one of the prerequisites for the 3D supports finding applications in tissue engineering. In this work, the potential use of the HAp/PCL scaffolds for bone tissue engineering applications was examined performing in vitro cell culture studies with preosteoblastic MC3T3-E1 cells in comparison with uncoated HAp derived from cuttlefish bone. MC3T3-E1 cells have been widely used as model cell to investigate various cell behaviors on scaffolds as bone substitute materials [31,32]. In a first set of experiments the effect of the HAp and HAp/PCL extracts in culture media was investigated by carrying out an indirect cellular viability assay, MTS test. Results (data not shown) indicated that materials do not exert any cytotoxic effect. The morphological and proliferative features observed of the cells exposed to the different concentrations of the extracts from HAp and HAp/PCL were similar to those observed for culture in tissue culture polystyrene plates. When the cells were seeded on the surface of the HAp and HAp/PCL scaffolds we observed that MC3T3-E1 adhered, proliferated and remained viable after 10 days of culture on both scaffolds as suggested by Live/Dead assay (Fig. 3). Fluorescence microscopy has shown that after 14 days of culture the cells proliferated on the surface and within the pores of both scaffolds. The cells on the HAp/PCL scaffold appeared to be denser compared with those on the HAp scaffold resulting in a partial or even complete closure of some pores. After 21 days of culture fibrillar structure of the extracellular matrix was seen on both scaffolds (see Fig. 5) and was confirmed with the SEM images (Fig. 2). Fluorescent micrographs of the HAp/PCL scaffold inner regions revealed that the penetration of cells occurred through the entire depth of the scaffold over the experimental period (21 days) (Fig. 6). In our opinion, this result is quite promising since it demonstrates that the scaffold possesses an adequate pore size and interconnectivity allowing the cell ingrowths surrounded by a sufficient diffusion of nutrients and oxygen. The SEM microscopy of the cell-seeded scaffolds also showed that most of the outer macropores of the HAp/PCL scaffold became sealed off by a continuous layer of cells. The cells spanned around the pore wall and formed an extracellular matrix. Analysis of DNA values showed that both scaffolds are suitable 3D supports for cellular proliferation. Despite the lower number of cells attached at the earliest time point the higher cellular proliferation occurred on the HAp/PCL scaffold compared to the porous HAp scaffold. The cellular differentiation was assessed by monitoring the ALP enzyme activity. Alkaline phosphatase (ALP) is a key enzyme in bone matrix vesicles that cleaves organic phosphate esters, thus supplying mineral nucleation sites with free phosphate ions [33–35]. ALP activity is generally considered an early stage marker for osteoblast phenotype and an important indicator of differentiation and mineralization [36]. Cell growth and expression of the osteoblastic phenotype are generally defined in three phases: 1) proliferation accompanied with the formation of the extracellular matrix; 2) matrix maturation accompanied by down-regulation of proliferation and up-regulation of ALP expression; 3) and mineralization marked by further decrease of proliferation and a decline of ALP activity [37]. The cells grown showed increasing amounts of ALP activity with the time of culture reaching a maximal value on day 14 of culture. An increase in bone specific ALP activity followed by a significant decrease at day 21 showed that the preosteoblasts seeded onto the scaffolds undergo a differentiation process into the direction of a mature phenotype. As already mentioned, analyses of the expression patterns of regulating genes and bonerelated proteins reveal three sequential stages during proliferation and


differentiation of MC3T3-E1 cells, including proliferation, bone matrix formation and maturation, and mineralization [38,39]. Choi et al. [39] found that ALP activity is highly expressed during the early matrix formation and maturation period, followed by a decrease in its activity, and during the mineralization period, the expression of osteocalcin is high. The synthesis of collagen as an extracellular matrix component by MC3T3-E1 cells was measured using hydroxyproline assay. Total collagen production within the HAp and the HAp/PCL scaffolds steadily increased with time in culture, in a statistically significant manner indicating the formation of a new extracellular matrix. At days 14 and 21 in culture a significant higher level of collagen production was observed within the HAp/PCL scaffold compared with the HAp scaffold suggesting that the PCL coating on the HAp scaffold may induce better cell attachment and consequently higher levels of collagen secretion. Immunofluorescence staining of the HAp/PCL sections revealed expression of collagen type I that was low on the third day of culture but increased on the third week of culture indicating again cell differentiation within the composite scaffolds. Based on these results, the HAp/PCL scaffold was shown to sustain MC3T3-E1 attachment and growth towards a mature and differentiated state. A positive effect of PCL coating on proliferation, differentiation and ECM deposition was observed compared with the uncoated HAp scaffold. Similar results were obtained recently by Kim at al. [28] performing an in vitro biological evaluation with human osteoblast-like MG-63 cells. Further studies need to be done to investigate the long-term degradation behavior of these composites in vitro. In vivo studies will be carried out as well to completely assess the biological performance on this scaffold. 5. Conclusions Natural aragonite from cuttlefish bone was hydrothermally transformed into hydroxyapatite (HAp) at 200 °C preserving the natural well interconnected porous structure. The obtained HAp scaffold was coated with a poly(ε-caprolactone) (PCL) using vacuum impregnation technique. Previous reported results have shown that the HAp/PCL composite scaffold possesses adequate porosity and quite good mechanical properties for being used in bone tissue engineering applications. In vitro cell culture studies showed that the scaffold is nontoxic and provides an adequate 3D support for the attachment, proliferation and differentiation of MC3T3-E1. Moreover, the proliferation and differentiation were more favorable on the PCL coated HAp scaffold with respect to the HAp scaffold itself. These results demonstrate the potential use of the HAp/PCL scaffolds in bone tissue engineering. Acknowledgments The financial support of the Ministry of Science, Education and Sports of the Republic of Croatia (Project 125-1252970-3005: “Bioceramic, Polymer and Composite Nanostructured Materials”), the Spanish Ministry project DPI2010-20399-C04-03 and the L'Oréal ADRIA-UNESCO national fellowship program for Women in Science is gratefully acknowledged. References [1] Y. Ohbayashi, M. Miyake, S. Nagahata, A long-term study of implanted artificial hydroxyapatite particles surrounding the carotid artery in adult dogs, Biomaterials 21 (5) (2000) 501–509. [2] A.R. Boccaccini, J.A. Roether, L.L. Hench, V. Maquet, R. Jerome, A composite approach to tissue engineering, Ceram. Eng. Sci. Proc. 23 (4) (2002) 805–816. [3] Y.M. Khan, D.S. Katti, C.T. Laurencin, Novel polymer-synthesized ceramic compositebased system for bone repair: an in vitro evaluation, J. Biomed. Mater. Res. A. 69 (4) (2004) 728–737. [4] M. Navarro, M.P. Ginebra, J.A. Planell, S. Zeppetelli, L. Ambrosio, Development and cell response of a new biodegradable composite scaffold for guided bone regeneration, J. Mater. Sci. Mater. Med. 15 (4) (2004) 419–422.

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